The applicants are authors or coauthors of two articles directed to the subject matter of the instant invention: (1) [applicants only] xe2x80x9cStudies of Endotoxin-Induced Decrease in Lipoprotein Lipase Activityxe2x80x9d, J. EXP. MED. 154 at 631-639 (September, 1981, published after Sep. 8, 1981), incorporated herein by reference; and (2) [co-authors with Phillip H. Pekala and M. Daniel Lane]: xe2x80x9cLipoprotein Lipase Suppression in 3T3-L1 Cells by an Endotoxin-Induced Mediator from Exudate Cellsxe2x80x9d, PROC. NAT""L. ACAD. SCI. 79 at 912-916 (February, 1982, published after Feb. 22, 1982), also incorporated herein by reference.
1. Field of the Invention
The present invention is directed generally to methods and associated materials for analysis of the effect and operation of invasive stimuli upon animal hosts, and in particular, is concerned with the mechanism and magnitude of the effect that such invasive stimuli may exert upon the activity of anabolic enzymes present in the host.
2. Description of the Prior Art
Several common physiological and biochemical derangement have been seen in various mammalian hosts responding to variety of invasive stimuli such as bacterial, viral and protozoan infections, as well as tumors and endotoxemia. For example, these responses include fever, leukocytosis, hyperlipidemia, reduced food intake and activity, and other modifications in muscle, white blood cell and liver metabolism. Recently, a hypertriglyceridemia in rabbits infected with a protozoan parasite, Trypanosoma brucei was reported by C. A. Rouser and A. Cerami, MOL. BIOCHEM. PARASITOL. 1 at 31-38 (1980). The reported hypertriglyceridemia was accompanied by a marked decrease in the activity of the enzyme lipoprotein lipase (LPL) in peripheral tissues.
LPL activity has been observed by others, and it has been noted that this condition has existed when the human bodywas in shock. See E. B. Man, et al, xe2x80x9cThe Lipids of Serum and Liver in Patients with Hepatic Diseasesxe2x80x9d, J. CLIN. INVEST. 24 at 623, et seq. (1945); See also John I. Gallin, et al, xe2x80x9cSerum Lipids in Infectionxe2x80x9d, N.ENGL. J. MED. 281 at 1081-1086 (Nov. 13, 1969); D. Farstchi, et al., xe2x80x9cEffects of Three Bacterial Infections on Serum Lipids of Rabbitsxe2x80x9d, J. BACTERIOL. 95 at 1615, et seq. (1968) S. E. Grossberg, et al., xe2x80x9cHyperlipaemia Following Viral Infection in the Chicken Embryo: A New Syndromexe2x80x9d, NATURE (London) 208 at 954, et seq. (1965); Robert L. Hirsch, et al., xe2x80x9cHyperlipidemia, Fatty Liver and Bromsulfophthalein Retention in Rabbits Injected Intravenously with Bacterial Endotoxinxe2x80x9d, J. LIPID. RES. 5 at 563-568 (1964); and Osamu Sakaguchi, et al., xe2x80x9cAlterations of Lipid Metabolism in Mice Injected with Endotoxinsxe2x80x9d, MICROBIOL. IMMUNOL. 23 (2) at 71-85 (1979); R. F. Kampschmidt, xe2x80x9cThe Activity of Partially Purified Leukocytic Endogeneous Meliator in Endotoxin-Resistant C3H/HeJ Micexe2x80x9d, J. LAB. CLIN. MED. 95 at 616, et seq. (1980); and Ralph F. Kampschmidt, xe2x80x9cLeukocytic Endogeneous Mediatorxe2x80x9d, J. RET. SOC. 23 (4) at 287-297 (1978).
While the existence of xe2x80x9cmediatorsxe2x80x9d was at least suspected, the effect, if any, that they had on general anabolic activity of energy storage cells was not known. The presentapplicants suspected that these xe2x80x9cmediatorsxe2x80x9d exerted a depressive effect upon the activity of certain anabolic enzymes, whose reduced activity was observed in instances where the host entered the condition of shock in response to invasion. Thus, the relationship of the mediator produced by endotoxin-stimulated peritoneal mouse exudate cells, upon endotoxin-sensitive and endotoxin insensitive mice alike, and the development through this investigation, of a reagent for measuring anabolic enzyme activity, was set forth in Ser. No. 299,932, and the further investigation of this system in conjunction with the 3T3 L1 xe2x80x9cpreadipocytexe2x80x9d model system, and the corresponding development of methods and associated materials for developing antibodies to the xe2x80x9cmediatorsxe2x80x9d as well as screening procedures for the identification and development of drugs capable of controlling the activity of these xe2x80x9cmediatorsxe2x80x9d was set forth in application Ser. No. 351,290. The work done to date indicates that a need exists for methodology andassociated diagnostic materials, to enable further investigationof the xe2x80x9cmediatorxe2x80x9d phenomenon to proceed, as well as to provide practical diagnostic tools useful in the treatment of the adversesequclae of infection and concomitant shock.
In accordance with a first aspect of the present invention, a method for preparing a mediator substance for use in assessing the state of anabolic enzymes in mammals, is disclosed, whichfinds particular utility in the instance where the mammals areundergoing invasive stimuli such as, viral agents, bacteria, protozoa, tumors, endotoxemia and others. In its simplest aspect, the method comprises gathering a sample of macrophage cells from amammal and incubating a portion of the macrophage cells with astimulator material associated with an invasive event. Forexample, the stimulator material may be endotoxin, in the instanceof endotoxemia, trypanosomes, in the instance of the above mentioned protozoan parasite Trypanosoma brucei, and others.
While the peritoneal exudate cells illustrated in our present and previous applications exemplify sources for themacrophage cells, it is to be understood that such cells may begathered from other than the peritoneal area, and that the present invention contemplates such variation within its scope.
The macrophage cells and the stimulator material are incubated as indicated, and thereafter, the macrophage cells areinduced to produce a mediator substance capable of supressing theactivity of the anabolic enzymes. Preferably, the inducement ofmediator production is accomplished during the incubation periodwhich may, for example, extend up to about 20 hours. The resulting medium may be appropriately treated to recover the mediator substance, and, for example, may be centrifuged, and the supernatant containing the mediator substance drawn off, or the mediator may be precipitated with a 40-60% solution of ammonium sulfate
As mentioned earlier, the mediator substance has a broad range of effects, including inhibitive effects that have beenobserved with respect to anabolic enzymes such as lipoproteinlipase (LPL), acetyl Coenzyme A carboxylase, fatty acid synthetase and the like. Also inhibitive effects have been found with red blood cell formation, as the mediator substance has been found tobe capable of inhibiting the growth and differentiation of erythroid committed cells, by the suppression of a number of growth and differentiation inducers, such as dimethylsulfoxide (DMSO),hexamethylene bisacetamide, butyric acid, hypoxanthine and thelike, as illustrated later on herein in specific examples.
A further embodiment of the present invention comprises a method for detecting various invasive stimuli by their capability of inhibiting the activity of one or more anabolic enzymes. In this method, a plurality of macrophage cell samples, may beprepared and selectively inoculated with a number of known stimulator materials, each characteristic in its effect upon differing anabolic factors. One of the macrophage samples may be inoculated with material from the presumed situs of the infective stimulus, and all samples may thereafter be incubated in accordance with the method described above. Thereafter, testing of each of the supernatants with the mediator substances derived fromthe known stimulator materials, would provide a comparativecontinuum for the identification of any invasive stimulus foundpresent. This testing method may utilize the 3T3 L1 cell system, for example, in the instance where lipoprotein lipase (LPL)activity is utilized as a parameter. Likewise, in the instancewhere red cell inducers are utilized, the Friend virus-transformed erythroleukemia cells may be inoculated and thereafter observed.See Friend, C., Sher, W. Holland J. G. and Sato, G. PROC. NATL.ACAD. SCI. 68, at 378-382; Marks, P. A., Rifkind, R. A., Terada, M., Ruben, R. C., Gazitt, Y. and Fibach, E. in ICN-UCLA Symposia on Molecular and Cellular Biology, Vol. X. xe2x80x9cHematopoietic Cell Differentiationxe2x80x9d. Ed. by D. W. Golde, M. J. Kline, D. Metcalf and C. F. Fox (Academi Press, New York), pp. 25-35 (1978). Naturally, other cellular systems may be utilized in the instance wherespecific activities may be appropriately observed, and the invention is not limited to the specific cellular systems set forth herein.
The invention includes methods for detecting the presence of samples of the various invasive stimuli in mammals by measuring mediator substance activity in the mammals. Thus, a number of mediatorsubstances may be prepared from the incubation of individual cell samples with known stimulator materials, and these mediator samples may thereafter be used to raise antibodies capable of specifically detecting the presence of the respective mediator substance. These antibodies may be prepared by known techniques, includingthe well known hybridoma technique for example, with fused mousespleen lymphocytes and myeloma, or by development in various animals such as rabbits, goats and other mammals. The known mediator samples and their antibodies may be appropriately labelled and utilized to test for the presence of the mediator substance in, for example, serum, as one may measure the degree of infection, and determine whether infection is increasing or abating, by observing the activity of the mediator substance therein. A variety of well known immunological techniques may be utilized in accordance with this aspect of the present invention, including single and double antibody techniques, utilizing detectible labels associated with either the known mediator substances, or their respective associated antibodies.
A further embodiment of the present invention relates to a method for preventing the occurrence of shock in mammals,comprising detecting the presence and shock promoting activity ofa mediator substance in the mammal, and thereafter administeringan antibody to the mediator substance, in an amount effective toprevent the development of shock in the mammal.
The invention particularly relates to a method of treating shock in humans, comprising administering to a human a shock-reducing amount of an antibody specific to a mediator substance.
Also, an assay system is disclosed and may be prepared for the screening of drugs potentially effective to inhibit thesynthesis or activity of the mediator substance. In the former instance, the effect of the test drug on the production of mediator by stimulated macrophages is determined, In the latter instance,a mediator substance may be introduced to cellular test systems, such as the 3T3 L1 cells, and the prospective drug may then be introduced to the resulting cell culture and the culture thereafter examined to observe any changes in mediator activity, either fromthe addition of the prospective drug alone, or the effect ofadded quantities of the known mediator substance.
A number of materials, compounds and agents have already been tested to determine their effect if any on mediator substanceproduction and activity. As discussed in further detail in thedescription, infra., only the steroid dexamethasone exhibited any inhibitory effect, and that effect appeared to be limited to theproduction of the mediator substance. Further agents, drugs, etc. can however be tested in the manner such as that employed withdexamethasone, and described herein.
The preparation of the mediator substance, and the determination of the importance of its activity, has resulted in the development of numerous avenues of diagnostic and therapeutic application. It is clear from the foregoing and following, thatthe detection of invasive stimuli may be made by the identification of the mediator substance, either directly or through the development of antibodies useful in immunological diagnosis.Further, these same antibodies may be utilized for direct treatment by control of mediator activity, to avert the development of shock in mammals, while the mediator substance may be utilized as screening agents in an assay system for the identification ofdrugs, agents and other substance (capable of neutralizing theadverse effects of the mediator substance, and thereby providing treatment of the adverse sequelae of infection.
Accordingly, it is a principal object of the present invention to provide a method for the preparation of a mediator substance exhibiting suppressive effects upon anabolic enzyme activity in mammals.
It is a further object of the present invention to provide a method for detecting the presence of a mediator substance in mammals in which invasive stimuli such as infection are suspected to be present.
It is a further object of the present invention to provide a method and associated assay system, for screening substance such as drugs, agents and the like, potentially effective incombating the adverse effects of the mediator substances in mammals.
It is a yet further object of the present invention to provide a method for the treatment of mammals to control the activity of said mediator substance so as to mitigate or avert the adverse consequences of their activity.
Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing description,which proceeds with reference to the following illustrativedrawings